Nitrite’s got to be cruel to be kind

The human pathogen Vibrio cholerae has puzzled the scientists since its discovery 150 years ago. Experts who studied the bacterium couldn't understand that the bacterium was not able to grow under anaerobic conditions despite it was equipped with an active metabolic machinery which should enable the organism to respire nitrate instead of oxygen, conditions that typically exist in the gastrointestinal tract. The common opinion was that the bacteria accumulates the intermediate product nitrite which inhibits further growth. The research group of Felipe Cava at The Laboratory for Molecular Infection Medicine Sweden studied this bacteria now under low-oxygen and different pH-conditions. Together with their colleagues in Boston, USA, the scientists at Umeå University discovered an elegant pH-dependent metabolic mechanism which permits the pathogen to switch to a resting mode with preserved viability. A smart strategy that provides competitive advantage against commensal bacteria to better colonize and infect the intestine. The group published the results now in the latest issue of Nature Microbiology 1 October 2018

It is for all known that certain actions can be misinterpreted as being negative, when in reality, they are intended to be positive. However, it is surprising that this popular saying can also be applied to bacteria like Vibrio cholerae, a human pathogen that causes deadly outbreaks of cholera disease, a very severe watery diarrhea affecting millions people every year.

To survive and proliferate in the absence of oxygen, many enteric pathogens as Vibrio cholerae can undergo anaerobic respiration within the host by respiring nitrate instead of oxygen. Respiratory nitrate reduction produces nitrite – a toxic agent – and thus, most nitrate reducing bacteria normally have additional enzymes to prevent nitrite accumulation. However, Vibrio cholerae accumulates nitrite and stops growth, increasing the belief that nitrate respiration is useless, or even detrimental, for this pathogen. Surprisingly scientists at Umeå University (Sweden) and their colleagues at Harvard University (USA), have now proved that rather than cruel, nitrite is a very kind molecule to Vibrio cholerae's life style.

"Nitrate reduction has been reported to be highly induced during infection. We were not convinced that this process could be negative for V. cholerae fitness", commented Felipe Cava.

Previous reports showed that, under anaerobic conditions, V. cholerae cultures supplemented with nitrate grew less than without nitrate, consistent with the belief that nitrite was toxic. Emilio Bueno and Felipe Cava wanted to go further and, in addition to growth, they tested the viability of bacteria. Surprisingly, they got an unexpected result that changed the project.

"I thought that there should be some kind of technical problem but I repeated the experiment 10 times with the same results: the viability and growth data were contradictory. We found that, although less grown, V. cholerae cultures supplemented with nitrate fully retained their viability whereas without nitrate they were largely dead", commented Bueno, postdoctoral fellow in the Cava-lab.

Given that these results proved that nitrite was not responsible of the killing, the team suspected that there was an intriguing mechanism behind this phenomenon.

"We found a mutant that survived in the absence of nitrate. This mutant was affected in its fermentative capacity, a pathway that in V. cholerae generates a strong acidification of the media. Therefore we reasoned that the acidity was the factor compromising Vibrio cholerae's viability while nitrite's seemingly toxicity was inducing a growth arrest state that ultimately prevented the cells to undergo fermentative suicide" explained Emilio Bueno.

Remarkably, the authors found that when the pH of the media was higher, Vibrio cholerae was perfectly capable to grow anaerobically with nitrate and hence, efficiently competed with other bacteria such as intestinal commensals. Given that V. cholerae colonizes the human intestine, – which is a rather anaerobic environment – these results might be particularly relevant during an infection.

The authors observed that these findings are not exclusive of V. cholerae. Similar results were obtained with Salmonella typhimurium, enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium.

"It seems that diverse enteric pathogens share the same pH-dependent mechanism to control population expansion and survival in anoxic environments with nitrate. Therefore our work might inspire the development of new strategies to control gastrointestinal infections and outbreaks. However, the impact of these discoveries might reach far beyond the context of an infection. Therefore it is important to study whether nitrate reducing bacteria can benefit from this metabolic switch in the free environment or when associated with other hosts", concludes Felipe Cava.